RESUMEN
The bioelectrical signatures associated with regeneration, wound healing, development, and cancer are changes in the polarization state of the cell that persist over long durations, and are mediated by ion channel activity. To identify physiologically relevant bioelectrical changes that occur during normal development of the sea urchin Lytechinus variegatus, we tested a range of ion channel inhibitors, and thereby identified SCH28080, a chemical inhibitor of the H(+)/K(+) ATPase (HKA), as an inhibitor of skeletogenesis. In sea urchin embryos, the primary mesodermal lineage, the PMCs, produce biomineral in response to signals from the ectoderm. However, in SCH28080-treated embryos, aside from randomization of the left-right axis, the ectoderm is normally specified and differentiated, indicating that the block to skeletogenesis observed in SCH28080-treated embryos is PMC-specific. HKA inhibition did not interfere with PMC specification, and was sufficient to block continuing biomineralization when embryos were treated with SCH28080 after the initiation of skeletogenesis, indicating that HKA activity is continuously required during biomineralization. Ion concentrations and voltage potential were abnormal in the PMCs in SCH28080-treated embryos, suggesting that these bioelectrical abnormalities prevent biomineralization. Our results indicate that this effect is due to the inhibition of amorphous calcium carbonate precipitation within PMC vesicles.
Asunto(s)
Exoesqueleto/química , Embrión no Mamífero/enzimología , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Morfogénesis/fisiología , Erizos de Mar/embriología , Animales , Cartilla de ADN/genética , Embrión no Mamífero/citología , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica/genética , Regulación Enzimológica de la Expresión Génica/genética , Imidazoles , Hibridación Fluorescente in Situ , Minerales/análisis , Reacción en Cadena de la Polimerasa , Erizos de Mar/enzimologíaRESUMEN
BACKGROUND: Nfkb1-deficient murine macrophages express higher levels of IFN-ß and IL-12 p40 following LPS stimulation than control macrophages, but the molecular basis for this phenomenon has not been completely defined. Nfkb1 encodes several gene products including the NF-κB subunit p50 and its precursor p105. p50 is derived from the N-terminal of 105, and p50 homodimers can exhibit suppressive activity when overexpressed. The C-terminal region of p105 is necessary for LPS-induced ERK activation and it has been suggested that ERK activity inhibits both IFN-ß and IL-12 p40 following LPS stimulation. However, the contributions of p50 and the C-terminal domain of p105 in regulating endogenous IFN-ß(Ifnb) and IL-12 p40 (Il12b) gene expression in macrophages following LPS stimulation have not been directly compared. METHODOLOGY/PRINCIPAL FINDINGS: We have used recombinant retroviruses to express p105, p50, and the C-terminal domain of p105 (p105ΔN) in Nfkb1-deficient murine bone marrow-derived macrophages at near endogenous levels. We found that both p50 and p105ΔN inhibited expression of Ifnb, and that inhibition of Ifnb by p105ΔN depended on ERK activation, because a mutant of p105ΔN (p105ΔNS930A) that lacks a key serine necessary to support ERK activation failed to inhibit. In contrast, only p105ΔN but not p50 inhibited Il12b expression. Surprisingly, p105ΔNS930A retained inhibitory activity for Il12b, indicating that ERK activation was not necessary for inhibition. The differential effects of p105ΔNS930A on Ifnb and Il12b expression inversely correlated with the function of one of its binding partners, c-Rel. This raised the possibility that p105ΔNS930A influences gene expression by interfering with the function of c-Rel. CONCLUSIONS: These results demonstrate that Nfkb1 exhibits multiple gene-specific inhibitory functions following TLR stimulation of murine macrophages.